Novel vaccine composition

ABSTRACT

An inactivated influenza virus preparation is described which comprises a haemagglutinin antigen stabilised in the absence of thiomersal, or at low levels of thiomersal, wherein the haemagglutinin is detectable by a SRD assay. The influenza virus preparation may comprise a micelle modifying excipient, for example α-tocopherol or a derivative thereof in a sufficient amount to stabilise the haemagglutinin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending applicationSer. No. 10/480,952, filed 22 Jun. 2004, which is a National Stage Entryof International Application No. PCT/EP02/05883, filed 29 May 2002, thecontents of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to novel influenza virus antigen preparations,methods for preparing them and their use in prophylaxis or therapy. Inparticular the invention relates to inactivated influenza vaccines whichare disrupted rather than whole virus vaccines and which are stable inthe absence of organomercurial preservatives. Moreover, the vaccinescontain haemagglutinin which is stable according to standard tests. Thevaccines can be administered by any route suitable for such vaccines,such as intramuscularly, subcutaneously, intradermally or mucosally e.g.intranasally.

BACKGROUND OF THE INVENTION

Influenza virus is one of the most ubiquitous viruses present in theworld, affecting both humans and livestock. The economic impact ofinfluenza is significant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the HI (haemagglutinin inhibition) assaydone in Example 10.

FIG. 2 shows the results of the HI (haemagglutinin inhibition) assaydone in Example 12.

SUMMARY OF THE INVENTION

The influenza virus is an RNA enveloped virus with a particle size ofabout 125 nm in diameter. It consists basically of an internalnucleocapsid or core of ribonucleic acid (RNA) associated withnucleoprotein, surrounded by a viral envelope with a lipid bilayerstructure and external glycoproteins. The inner layer of the viralenvelope is composed predominantly of matrix proteins and the outerlayer mostly of host-derived lipid material. The surface glycoproteinsneuraminidase (NA) and haemagglutinin (HA) appear as spikes, 10 to 12 nmlong, at the surface of the particles. It is these surface proteins,particularly the haemagglutinin, that determine the antigenicspecificity of the influenza subtypes.

DETAILED DESCRIPTION OF THE INVENTION

Currently available influenza vaccines are either inactivated or liveattenuated influenza vaccine. Inactivated flu vaccines are composed ofthree possible forms of antigen preparation: inactivated whole virus,sub-virions where purified virus particles are disrupted with detergentsor other reagents to solubilise the lipid envelope (so-called “split”vaccine) or purified HA and NA (subunit vaccine). These inactivatedvaccines are given intramuscularly (i.m.) or intranasally (i.n.). Thereis no commercially available live attenuated vaccine.

Influenza vaccines, of all kinds, are usually trivalent vaccines. Theygenerally contain antigens derived from two influenza A virus strainsand one influenza B strain. A standard 0.5 ml injectable dose in mostcases contains 15 μg of haemagglutinin antigen component from eachstrain, as measured by single radial immunodiffusion (SRD) (J. M. Woodet al.: An improved single radial immunodiffusion technique for theassay of influenza haemagglutinin antigen: adaptation for potencydetermination of inactivated whole virus and subunit vaccines. J. Biol.Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborativestudy of single radial diffusion and immunoelectrophoresis techniquesfor the assay of haemagglutinin antigen of influenza virus. J. Biol.Stand. 9 (1981) 317-330).

The influenza virus strains to be incorporated into influenza vaccineeach season are determined by the World Health Organisation incollaboration with national health authorities and vaccinemanufacturers.

Typical influenza epidemics cause increases in incidence of pneumoniaand lower respiratory disease as witnessed by increased rates ofhospitalisation or mortality. The elderly or those with underlyingchronic diseases are most likely to experience such complications, butyoung infants also may suffer severe disease. These groups in particulartherefore need to be protected.

Current efforts to control the morbidity and mortality associated withyearly epidemics of influenza are based on the use of intramuscularlyadministered inactivated influenza vaccines. The efficacy of suchvaccines in preventing respiratory disease and influenza complicationsranges from 75% in healthy adults to less than 50% in the elderly.

Standards are applied internationally to measure the efficacy ofinfluenza vaccines. The European Union official criteria for aneffective vaccine against influenza are set out in the table below.Theoretically, to meet the European Union requirements, an influenzavaccine has to meet only one of the criteria in the table, for allstrains of influenza included in the vaccine. However in practice, atleast two or all three of the criteria will need to be met for allstrains, particularly for a new vaccine such as a new vaccine fordelivery via a different route. Under some circumstances two criteriamay be sufficient. For example, it may be acceptable for two of thethree criteria to be met by all strains while the third criterion is metby some but not all strains (e.g. two out of three strains). Therequirements are different for adult populations (18-60 years) andelderly populations (>60 years).

18-60 years >60 years Seroconversion rate* >40% >30% Conversionfactor** >2.5 >2.0 Protection rate*** >70% >60% *Seroconversion rate isdefined as the percentage of vaccinees who have at least a 4-foldincrease in serum haemagglutinin inhibition (HI) titres aftervaccination, for each vaccine strain. **Conversion factor is defined asthe fold increase in serum HI geometric mean titres (GMTs) aftervaccination, for each vaccine strain. ***Protection rate is defined asthe percentage of vaccinees with a serum HI titre equal to or greaterthan 1:40 after vaccination (for each vaccine strain) and is normallyaccepted as indicating protection.

For a novel flu vaccine to be commercially useful it will not only needto meet those standards, but also in practice it will need to be atleast as efficacious as the currently available injectable vaccines. Itwill also need to be commercially viable in terms of the amount ofantigen and the number of administrations required.

The current commercially available influenza vaccines are either splitor subunit injectable vaccines. These vaccines are prepared bydisrupting the virus particle, generally with an organic solvent or adetergent, and separating or purifying the viral proteins to varyingextents. Split vaccines are prepared by fragmentation of whole influenzavirus, either infectious or inactivated, with solubilizingconcentrations of organic solvents or detergents and subsequent removalof the solubilizing agent and some or most of the viral lipid material.Split vaccines generally contain contaminating matrix protein andnucleoprotein and sometimes lipid, as well as the membrane envelopeproteins. Split vaccines will usually contain most or all of the virusstructural proteins although not necessarily in the same proportions asthey occur in the whole virus. Subunit vaccines on the other handconsist essentially of highly purified viral surface proteins,haemagglutinin and neuraminidase, which are the surface proteinsresponsible for eliciting the desired virus neutralising antibodies uponvaccination.

Many vaccines which are currently available require a preservative toprevent deterioration. A frequently used preservative is thimerosalwhich is a mercury-containing compound. Some public concerns have beenexpressed about the effects of mercury containing compounds. There is nosurveillance system in place to detect the effects of low to moderatedoses of organomercurials on the developing nervous system, and specialstudies of children who have received high doses of organomercurialswill take several years to complete. Certain commentators have stressedthat the potential hazards of thimerosal-containing vaccines should notbe overstated (Offit; P. A. JAMA Vol. 283; No:16). Nevertheless, itwould be advantageous to find alternative methods for the preparation ofvaccines to replace the use of thiomerosal in the manufacturing process.There is thus a need to develop vaccines which are thimerosal-free, inparticular vaccines like influenza vaccines which are recommended, atleast for certain population groups, on an annual basis.

It has been standard practice to date to employ a preservative forcommercial inactivated influenza vaccines, during theproduction/purification process and/or in the final vaccine. Thepreservative is required to prevent microorganisms from growing throughthe various stages of purification. For egg-derived influenza vaccines,thiomersal is typically added to the raw allantoic fluid and may also beadded a second time during the processing of the virus. Thus there willbe residual thiomersal present at the end of the process, and this mayadditionally be adjusted to a desirable preservative concentration inthe final vaccine, for example to a concentration of around 100 μg/ml.

A side-effect of the use of thiomersal as a preservative in flu vaccinesis a stabilisation effect. The thiomersal in commercial flu vaccinesacts to stabilise the HA component of the vaccine, in particular but notexclusively HA of B strain influenza. Certain A strain haemagglutininse.g. H3 may also require stabilisation. Therefore, although it may bedesirable to consider removing thiomersal from influenza vaccines, or atleast reducing the concentration of the thiomersal in the final vaccine,there is a problem to overcome in that, without thiomersal, the HA willnot be sufficiently stable.

It has been discovered in the present invention that it is possible tostabilise HA in inactivated influenza preparations using alternativereagents that do not contain organomercurials. The HA remains stabilisedsuch that it is detectable over time by quantitative standard methods,in particular SRD, to a greater extent than a non-stabilised antigenpreparation produced by the same method but without stabilisingexcipient. The SRD method is performed as described hereinabove.Importantly, the HA remains stabilised for up to 12 months which is thestandard required of a final flu vaccine. In a first aspect the presentinvention provides an inactivated influenza virus preparation comprisinga haemagglutinin antigen stabilised in the absence of thiomersal, or atlow levels of thiomersal, wherein the haemagglutinin is detectable by aSRD assay.

Low levels of thiomersal are those levels at which the stability of HAderived from influenza B is reduced, such that a stabilising excipientis required for stabilised HA. Low levels of thiomersal are generally 5μg/ml or less.

Generally, stabilised HA refers to HA which is detectable over time byquantitative standard methods, in particular SRD, to a greater extentthan a non-stabilised antigen preparation produced by the same methodbut without any stabilising excipient. Stabilisation of HA preferablymaintains the activity of HA substantially constant over a one yearperiod. Preferably, stabilisation allows the vaccine comprising HA toprovide acceptable protection after a 6 month storage period, morepreferably a one year period.

Suitably, stabilisation is carried out by a stabilising excipient,preferably a micelle modifying excipient. A micelle modifying excipientis generally an excipient that may be incorporated into a micelle formedby detergents used in, or suitable for, solubilising the membraneprotein HA, such as the detergents Tween 80, Triton X100 anddeoxycholate, individually or in combination.

Without wishing to be constrained by theory, it is believed that theexcipients work to stabilise HA by interaction with the lipids,detergents and/or proteins in the final preparation. Mixed micelles ofexcipient with protein and lipid may be formed, such as micelles ofTween and deoxycholate with residual lipids and/or Triton X-100. It isthought that surface proteins are kept solubilised by those complexmicelles. Preferably, protein aggregation is limited by charge repulsionof micelles containing suitable excipients, such as micelles containingnegatively charged detergents.

Suitable micelle modifying excipients include: positively, negatively orzwitterionic charged amphiphilic molecules such as alkyl sulfates, oralkyl-aryl-sulfates; non-ionic amphiphilic molecules such as alkylpolyglycosides or derivatives thereof, such as Plantacare® (availablefrom Henkel KGaA), or alkyl alcohol poly alkylene ethers or derivativesthereof such as Laureth-9.

Preferred excipients are α-tocopherol, or derivatives of α-tocopherolsuch as α-tocopherol succinate. Other preferred tocopherol derivativesfor use in the invention include D-α tocopherol, D-δ tocopherol, D-γtocopherol and DL-α-tocopherol. Preferred derivatives of tocopherolsthat may be used include acetates, succinates, phosphoric acid esters,formiates, propionates, butyrates, sulfates and gluconates.Alpha-tocopherol succinate is particularly preferred. The α-tocopherolor derivative is present in an amount sufficient to stabilise thehaemagglutinin.

Other suitable excipients may be identified by methods standard in theart, and tested for example using the SRD method for stability analysisas described herein.

In a preferred aspect the invention provides an influenza virus antigenpreparation comprising at least one stable influenza B strainhaemagglutinin antigen.

The invention provides in a further aspect a method for preparing astable haemagglutinin antigen which method comprises purifying theantigen in the presence of a stabilising micelle modifying excipient,preferably α-tocopherol or a derivative thereof such as α-tocopherolsuccinate.

Further provided by the invention are vaccines comprising the antigenpreparations described herein and their use in a method for prophylaxisof influenza infection or disease in a subject which method comprisesadministering to the subject a vaccine according to the invention.

The vaccine may be administered by any suitable delivery route, such asintradermal, mucosal e.g. intranasal, oral, intramuscular orsubcutaneous. Other delivery routes are well known in the art.

Intradermal delivery is preferred. Any suitable device may be used forintradermal delivery, for example short needle devices such as thosedescribed in U.S. Pat. No. 4,886,499, U.S. Pat. No. 5,190,521, U.S. Pat.No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No. 4,270,537, U.S.Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat. No. 5,417,662.Intradermal vaccines may also be administered by devices which limit theeffective penetration length of a needle into the skin, such as thosedescribed in WO99/34850 and EP1092444, incorporated herein by reference,and functional equivalents thereof. Also suitable are jet injectiondevices which deliver liquid vaccines to the dermis via a liquid jetinjector or via a needle which pierces the stratum corneum and producesa jet which reaches the dermis. Jet injection devices are described forexample in U.S. Pat. No. 5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat.No. 5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat. No. 5,649,912, U.S.Pat. No. 5,569,189, U.S. Pat. No. 5,704,911, U.S. Pat. No. 5,383,851,U.S. Pat. No. 5,893,397, U.S. Pat. No. 5,466,220, U.S. Pat. No.5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat.No. 5,064,413, U.S. Pat. No. 5,520, 639, U.S. Pat. No. 4,596,556, U.S.Pat. No. 4,790,824, U.S. Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO97/37705 and WO 97/13537. Also suitable are ballistic powder/particledelivery devices which use compressed gas to accelerate vaccine inpowder form through the outer layers of the skin to the dermis.Additionally, conventional syringes may be used in the classical mantouxmethod of intradermal administration. However, the use of conventionalsyringes requires highly skilled operators and thus devices which arecapable of accurate delivery without a highly skilled user arepreferred.

The invention thus provides a method for the prophylaxis of influenzainfection or disease in a subject which method comprises administeringto the subject intradermally an influenza vaccine according to theinvention.

The invention also extends to intradermal devices in combination with avaccine according to the present invention, in particular with devicesdisclosed in WO99/34850 or EP1092444, for example.

Also provided is the use of a micelle modifying excipient, preferablyα-tocopherol or a derivative thereof as a haemagglutinin stablilser inthe manufacture of an influenza vaccine.

The invention applies particularly but not exclusively to thestabilisation of B strain influenza haemagglutinin.

Preferably the stabilised HA of the present invention is stable for 6months, more preferably 12 months.

Preferably the α-tocopherol is in the form of an ester, more preferablythe succinate or acetate and most preferably the succinate.

Preferred concentrations for the α-tocopherol or derivative are between1 μg/ml-10 mg/ml, more preferably between 10 μg/ml-500 μg/ml.

The vaccine according to the invention generally contains both A and Bstrain virus antigens, typically in a trivalent composition of two Astrains and one B strain. However, divalent and monovalent vaccines arenot excluded. Monovalent vaccines may be advantageous in a pandemicsituation, for example, where it is important to get as much vaccineproduced and administered as quickly as possible.

The non-live flu antigen preparation for use in the invention may beselected from the group consisting of split virus antigen preparations,subunit antigens (either recombinantly expressed or prepared from wholevirus), inactivated whole virus which may be chemically inactivated withe.g. formaldehyde, β-propiolactone or otherwise inactivated e.g. U.V. orheat inactivated. Preferably the antigen preparation is either a splitvirus preparation, or a subunit antigen prepared from whole virus,particularly by a splitting process followed by purification of thesurface antigen. Most preferred are split virus preparations.

Preferably the concentration of haemagglutinin antigen for each strainof the influenza virus preparation is 1-1000 μg per ml, more preferably3-300 μg per ml and most preferably about 30 μg per ml, as measured by aSRD assay.

The vaccine according to the invention may further comprise an adjuvantor immunostimulant such as but not limited to detoxified lipid A fromany source and non-toxic derivatives of lipid A, saponins and otherreagents capable of stimulating a TH1 type response.

It has long been known that enterobacterial lipopolysaccharide (LPS) isa potent stimulator of the immune system, although its use in adjuvantshas been curtailed by its toxic effects. A non-toxic derivative of LPS,monophosphoryl lipid A (MPL), produced by removal of the corecarbohydrate group and the phosphate from the reducing-end glucosamine,has been described by Ribi et al (1986, Immunology andImmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, p407-419) and has the following structure:

A further detoxified version of MPL results from the removal of the acylchain from the 3-position of the disaccharide backbone, and is called3-O-Deacylated monophosphoryl lipid A (3D-MPL). It can be purified andprepared by the methods taught in GB 2122204B, which reference alsodiscloses the preparation of diphosphoryl lipid A, and 3-O-deacylatedvariants thereof.

A preferred form of 3D-MPL is in the form of an emulsion having a smallparticle size less than 0.2 μm in diameter, and its method ofmanufacture is disclosed in WO 94/21292. Aqueous formulations comprisingmonophosphoryl lipid A and a surfactant have been described inWO9843670A2.

The bacterial lipopolysaccharide derived adjuvants to be formulated inthe compositions of the present invention may be purified and processedfrom bacterial sources, or alternatively they may be synthetic. Forexample, purified monophosphoryl lipid A is described in Ribi et al 1986(supra), and 3-O-Deacylated monophosphoryl or diphosphoryl lipid Aderived from Salmonella sp. is described in GB 2220211 and U.S. Pat. No.4,912,094. Other purified and synthetic lipopolysaccharides have beendescribed (Hilgers et al., 1986, Int. Arch. Allergy. Immunol.,79(4):392-6; Hilgers et al., 1987, Immunology, 60(1):141-6; and EP 0 549074 B1). A particularly preferred bacterial lipopolysaccharide adjuvantis 3D-MPL.

Accordingly, the LPS derivatives that may be used in the presentinvention are those immunostimulants that are similar in structure tothat of LPS or MPL or 3D-MPL. In another aspect of the present inventionthe LPS derivatives may be an acylated monosaccharide, which is asub-portion to the above structure of MPL.

Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. A reviewof the biological and pharmacological activities of saponins.Phytomedicine vol 2 pp 363-386). Saponins are steroid or triterpeneglycosides widely distributed in the plant and marine animal kingdoms.Saponins are noted for forming colloidal solutions in water which foamon shaking, and for precipitating cholesterol. When saponins are nearcell membranes they create pore-like structures in the membrane whichcause the membrane to burst. Haemolysis of erythrocytes is an example ofthis phenomenon, which is a property of certain, but not all, saponins.

Saponins are known as adjuvants in vaccines for systemic administration.The adjuvant and haemolytic activity of individual saponins has beenextensively studied in the art (Lacaille-Dubois and Wagner, supra). Forexample, Quil A (derived from the bark of the South American treeQuillaja Saponaria Molina), and fractions thereof, are described in U.S.Pat. No. 5,057,540 and “Saponins as vaccine adjuvants”, Kensil, C. R.,Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279B1. Particulate structures, termed Immune Stimulating Complexes(ISCOMS), comprising fractions of Quil A are haemolytic and have beenused in the manufacture of vaccines (Morein, B., EP 0 109 942 B1; WO96/11711; WO 96/33739). The haemolytic saponins QS21 and QS17 (HPLCpurified fractions of Quil A) have been described as potent systemicadjuvants, and the method of their production is disclosed in U.S. Pat.No. 5,057,540 and EP 0 362 279 B1. Other saponins which have been usedin systemic vaccination studies include those derived from other plantspecies such as Gypsophila and Saponaria (Bomford et al., Vaccine,10(9):572-577, 1992).

An enhanced system involves the combination of a non-toxic lipid Aderivative and a saponin derivative particularly the combination of QS21and 3D-MPL as disclosed in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol as disclosed inWO 96/33739.

A particularly potent adjuvant formulation involving QS21 and 3D-MPL inan oil in water emulsion is described in WO 95/17210 and is a preferredformulation.

Accordingly in one embodiment of the present invention there is provideda vaccine comprising an influenza antigen preparation of the presentinvention adjuvanted with detoxified lipid A or a non-toxic derivativeof lipid A, more preferably adjuvanted with a monophosphoryl lipid A orderivative thereof.

Preferably the vaccine additionally comprises a saponin, more preferablyQS21.

Preferably the formulation additionally comprises an oil in wateremulsion. The present invention also provides a method for producing avaccine formulation comprising mixing an antigen preparation of thepresent invention together with a pharmaceutically acceptable excipient,such as 3D-MPL.

The vaccines according to the invention may further comprise at leastone surfactant which may be in particular a non-ionic surfactant.Suitable non-ionic surfactants are selected from the group consisting ofthe octyl- or nonylphenoxy polyoxyethanols (for example the commerciallyavailable Triton™ series), polyoxyethylene sorbitan esters (Tween™series) and polyoxyethylene ethers or esters of general formula (I):

HO(CH₂CH₂O)_(n)-A-R   (I)

wherein n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or phenylC₁₋₅₀ alkyl; and combinations of two or more of these.

Preferred surfactants falling within formula (I) are molecules in whichn is 4-24, more preferably 6-12, and most preferably 9; the R componentis C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂ alkyl.

Octylphenoxy polyoxyethanols and polyoxyethylene sorbitan esters aredescribed in “Surfactant systems” Eds: Attwood and Florence (1983,Chapman and Hall). Octylphenoxy polyoxyethanols (the octoxynols),including t-octylphenoxypolyethoxyethanol (Triton X-100™) are alsodescribed in Merck Index Entry 6858 (Page 1162, 12^(th) Edition, Merck &Co. Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3). Thepolyoxyethylene sorbitan esters, including polyoxyethylene sorbitanmonooleate (Tween 80™) are described in Merck Index Entry 7742 (Page1308, 12^(th) Edition, Merck & Co. Inc., Whitehouse Station, N.J., USA;ISBN 0911910-12-3). Both may be manufactured using methods describedtherein, or purchased from commercial sources such as Sigma Inc.

Particularly preferred non-ionic surfactants include Triton X-45,t-octylphenoxy polyethoxyethanol (Triton X-100), Triton X-102, TritonX-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57, TritonN-101, Triton N-128, Breij 35, polyoxyethylene-9-lauryl ether (laureth9) and polyoxyethylene-9-stearyl ether (steareth 9). Triton X-100 andlaureth 9 are particularly preferred. Also particularly preferred is thepolyoxyethylene sorbitan ester, polyoxyethylene sorbitan monooleate(Tween 80™).

Further suitable polyoxyethylene ethers of general formula (I) areselected from the following group: polyoxyethylene-8-stearyl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether.

Alternative terms or names for polyoxyethylene lauryl ether aredisclosed in the CAS registry. The CAS registry number ofpolyoxyethylene-9 lauryl ether is: 9002-92-0. Polyoxyethylene etherssuch as polyoxyethylene lauryl ether are described in the Merck index(12^(th) ed: entry 7717, Merck & Co. Inc., Whitehouse Station, N.J.,USA; ISBN 0911910-12-3). Laureth 9 is formed by reacting ethylene oxidewith dodecyl alcohol, and has an average of nine ethylene oxide units.

Two or more non-ionic surfactants from the different groups ofsurfactants described may be present in the vaccine formulationdescribed herein. In particular, a combination of a polyoxyethylenesorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80™)and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton) X-100™is preferred. Another particularly preferred combination of non-ionicsurfactants comprises laureth 9 plus a polyoxyethylene sorbitan ester oran octoxynol or both.

Non-ionic surfactants such as those discussed above have preferredconcentrations in the final vaccine composition as follows:polyoxyethylene sorbitan esters such as Tween 80™: 0.01 to 1%, mostpreferably about 0.1% (w/v); octyl- or nonylphenoxy polyoxyethanols suchas Triton X-100™ or other detergents in the Triton series: 0.001 to0.1%, most preferably 0.005 to 0.02% (w/v); polyoxyethylene ethers ofgeneral formula (I) such as laureth 9: 0.1 to 20%, preferably 0.1 to 10%and most preferably 0.1 to 1% or about 0.5% (w/v).

For certain vaccine formulations, other vaccine components may beincluded in the formulation. As such the formulations of the presentinvention may also comprise a bile acid or a derivative thereof, inparticular in the form of a salt. These include derivatives of cholicacid and salts thereof, in particular sodium salts of cholic acid orcholic acid derivatives. Examples of bile acids and derivatives thereofinclude cholic acid, deoxycholic acid, chenodeoxycholic acid,lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid andderivatives such as glyco-, tauro-, amidopropyl-1-propanesulfonic-,amidopropyl-2-hydroxy-1-propanesulfonic derivatives of theaforementioned bile acids, or N,N-bis (3Dgluconoamidopropyl)deoxycholamide. A particularly preferred example is sodium deoxycholate(NaDOC) which may be present in the final vaccine dose.

Also provided by the invention are pharmaceutical kits comprising avaccine administration device filled with a vaccine according to theinvention. Such administration devices include but are not limited toneedle devices, liquid jet devices, powder devices, and spray devices(for intranasal use).

The influenza virus antigen preparations according to the invention maybe derived from the conventional embryonated egg method, or they may bederived from any of the new generation methods using tissue culture togrow the virus or express recombinant influenza virus surface antigens.Suitable cell substrates for growing the virus include for example dogkidney cells such as MDCK or cells from a clone of MDCK, MDCK-likecells, monkey kidney cells such as AGMK cells including Vero cells,suitable pig cell lines, or any other mammalian cell type suitable forthe production of influenza virus for vaccine purposes. Suitable cellsubstrates also include human cells e.g. MRC-5 cells. Suitable cellsubstrates are not limited to cell lines; for example primary cells suchas chicken embryo fibroblasts are also included.

The influenza virus antigen preparation may be produced by any of anumber of commercially applicable processes, for example the split fluprocess described in patent no. DD 300 833 and DD 211 444, incorporatedherein by reference. Traditionally split flu was produced using asolvent/detergent treatment, such as tri-n-butyl phosphate, ordiethylether in combination with Tween™ (known as “Tween-ether”splitting) and this process is still used in some production facilities.Other splitting agents now employed include detergents or proteolyticenzymes or bile salts, for example sodium deoxycholate as described inpatent no. DD 155 875, incorporated herein by reference. Detergents thatcan be used as splitting agents include cationic detergents e.g. cetyltrimethyl ammonium bromide (CTAB), other ionic detergents e.g.laurylsulfate, taurodeoxycholate, or non-ionic detergents such as theones described above including Triton X-100 (for example in a processdescribed in Lina et al, 2000, Biologicals 28, 95-103) and Triton N-101,or combinations of any two or more detergents.

The preparation process for a split vaccine will include a number ofdifferent filtration and/or other separation steps such asultracentrifugation, ultrafiltration, zonal centrifugation andchromatography (e.g. ion exchange) steps in a variety of combinations,and optionally an inactivation step, e.g., with heat, formaldehyde orβ-propiolactone or U.V. which may be carried out before or aftersplitting. The splitting process may be carried out as a batch,continuous or semi-continuous process.

Preferred split flu vaccine antigen preparations according to theinvention comprise a residual amount of Tween 80 and/or Triton X-100remaining from the production process, although these may be added ortheir concentrations adjusted after preparation of the split antigen.Preferably both Tween 80 and Triton X-100 are present. The preferredranges for the final concentrations of these non-ionic surfactants inthe vaccine dose are:

-   Tween 80: 0.01 to 1%, more preferably about 0.1% (v/v)-   Triton X-100: 0.001 to 0.1 (% w/v), more preferably 0.005 to 0.02%    (w/v).

Alternatively the influenza virus antigen preparations according to theinvention may be derived from a source other than the live influenzavirus, for example the haemagglutinin antigen may be producedrecombinantly.

The invention will now be further described in the following,non-limiting examples.

EXAMPLES Example 1 Preparation of Influenza Virus Antigen PreparationUsing α-Tocopherol Succinate as a Stabiliser for a Preservative-FreeVaccine (Thiomersal-Reduced Vaccine)

Monovalent split vaccine was prepared according to the followingprocedure.

Preparation of Virus Inoculum

On the day of inoculation of embryonated eggs a fresh inoculum isprepared by mixing the working seed lot with a phosphate buffered salinecontaining gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25μg/ml. (virus strain-dependent). The virus inoculum is kept at 2-8° C.

Inoculation of Embryonated Eggs

Nine to eleven day old embryonated eggs are used for virus replication.Shells are decontaminated. The eggs are inoculated with 0.2 ml of thevirus inoculum. The inoculated eggs are incubated at the appropriatetemperature (virus strain-dependent) for 48 to 96 hours. At the end ofthe incubation period, the embryos are killed by cooling and the eggsare stored for 12-60 hours at 2-8° C.

Harvest

The allantoic fluid from the chilled embryonated eggs is harvested.Usually, 8 to 10 ml of crude allantoic fluid is collected per egg.

Concentration and Purification of Whole Virus From Allantoic Fluid

1. Clarification

The harvested allantoic fluid is clarified by moderate speedcentrifugation (range: 4000-14000 g).

2. Adsorption Step

To obtain a CaHPO₄ gel in the clarified virus pool, 0.5 mol/L Na₂HPO₄and 0.5 mol/L CaCl₂ solutions are added to reach a final concentrationof CaHPO₄ of 1.5 g to 3.5 g CaHPO₄/litre depending on the virus strain.

After sedimentation for at last 8 hours, the supernatant is removed andthe sediment containing the influenza virus is resolubilised by additionof a 0.26 mol/L EDTA-Na₂ solution, dependent on the amount of CaHPO₄used.

3. Filtration

The resuspended sediment is filtered on a 6 μm filter membrane.

4. Sucrose Gradient Centrifugation

The influenza virus is concentrated by isopycnic centrifugation in alinear sucrose gradient (0.55% (w/v)) containing 100 μg/ml Thiomersal.The flow rate is 8-15 litres/hour.

At the end of the centrifugation, the content of the rotor is recoveredby four different fractions (the sucrose is measured in arefractometer):

fraction 1 55-52% sucrose fraction 2 approximately 52-38% sucrosefraction 3 38-20% sucrose* fraction 4 20-0% sucrose *virusstrain-dependent: fraction 3 can be reduced to 15% sucrose.

For further vaccine preparation, only fractions 2 and 3 are used.

Fraction 3 is washed by diafiltration with phosphate buffer in order toreduce the sucrose content to approximately below 6%. The influenzavirus present in this diluted fraction is pelleted to remove solublecontaminants.

The pellet is resuspended and thoroughly mixed to obtain a homogeneoussuspension. Fraction 2 and the resuspended pellet of fraction 3 arepooled and phosphate buffer is added to obtain a volume of approximately40 litres. This product is the monovalent whole virus concentrate.

5. Sucrose Gradient Centrifugation With Sodium Deoxycholate

The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v) and Tocopherol succinate is added for B-strain-viruses up to 0.5mM. The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v) andis strain dependent. The flow rate is 8-15 litres/hour.

At the end of the centrifugation, the content of the rotor is recoveredby three different fractions (the sucrose is measured in arefractometer) Fraction 2 is used for further processing. Sucrosecontent for fraction limits (47-18%) varies according to strains and isfixed after evaluation:

6. Sterile Filtration

The split virus fraction is filtered on filter membranes ending with a0.2 μm membrane. Phosphate buffer containing 0.025% (w/v) Tween 80 and(for B strain viruses) 0.5 mM Tocopherol succinate is used for dilution.The final volume of the filtered fraction 2 is 5 times the originalfraction volume.

7. Inactivation

The filtered monovalent material is incubated at 22±2° C. for at most 84hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffer containing 0.025% (w/v). Tween 80 is thenadded in order to reduce the total protein content down to max. 250μg/ml. For B strain viruses, a phosphate buffered saline containing0.025% (w/v) Tween 80 and 0.25 mM Tocopherol succinate is applied fordilution to reduce the total protein content down to 250 μg/ml.Formaldehyde is added to a final concentration of 50 μg/ml and theinactivation takes place at 20° C.±2° C. for at least 72 hours.

8. Ultrafiltration

The inactivated split virus material is concentrated at least 2 fold ina ultrafiltration unit, equipped with cellulose acetate membranes with20 kDa MWCO. The Material is subsequently washed with phosphate buffercontaining 0.025% (w/v) Tween 80 and following with phosphate bufferedsaline containing 0.01% (w/v) Tween. For B strain virus a phosphatebuffered saline containing 0.01% (w/v) Tween 80 and 0.1 mM Tocopherolsuccinate is used for washing.

9. Final Sterile Filtration

The material after ultrafiltration is filtered on filter membranesending with a 0.2 μm membrane. Filter membranes are rinsed and thematerial is diluted if necessary such that the protein concentrationdoes not exceed 500 μg/ml with phosphate buffered saline containing0.01% (w/v) Tween 80 and (for B strain viruses) 0.1 mM Tocopherolsuccinate.

10. Storage

The monovalent final bulk is stored at 2-8° C. for a maximum of 18months.

Stability

TABLE 1 Comparison of time dependent HA content (μg/ml) measured by SRDin monovalent final bulks. After 4 weeks 6 month 12 month at StrainStabiliser production at 30° C. at 2-8° C. 2-8° C. B/Yamanashi/166/98Tocopherylsuccinate 169 139 172 ND (residual mercury 3 (82%) (>100%) μg/ml) B/Yamanashi/166/98 Thiomersal 192 160 186 178 (108 μg/ml) (83%)(97%)  (93%) B/Yamanashi/166/98 None 191 122 175 154 (residual mercury 3(60%) (92%)  (81%) μg/ml) B/Johannesburg/5/99 Tocopherylsuccinate 166183 158 179 (residual mercury 4 (>100%)  (95%) (>100%) μg/ml)B/Johannesburg/5/99 Tocopherylsuccinate 167 179 158 178 (residualmercury 4 (>100%)  (95%) (>100%) μg/ml) B/Johannesburg/5/99Tocopherylsuccinate 144 151 130 145 (residual mercury 3 (>100%)  (90%)(>100%) μg/ml) B/Johannesburg/5/99* Thiomersal 159 ND 172 154 (>100%)  (97%) B/Johannesburg/5/99** None 169 107 153 ON (63%) (90%) *producedaccording to licensed FLUARIX ™, **produced according to example 1without Tocopherylsuccinate, ON: Ongoing, ND not determined

Example 2 Preparation of Influenza Vaccine Using α-Tocopherol Succinateas a Stabiliser for a Thiomersal-Reduced Vaccine

Monovalent final bulks of three strains, A/New Caldonia/20/99 (H1N1)IVR-116, A/Panama/2007/99 (H3N2) Resvir-17 and B/Yamanashi/166/98 wereproduced according to the method described in Example 1.

Pooling

The appropriate amount of monovalent final bulks was pooled to a finalHA-concentration of 30 μg/ml for A/New Caldonia/20/99 (H1N1) IVR-116,A/Panama/2007/99 (H3N2) Resvir-17, respectively and of 39 μg/ml forB/Yamanashi/166/98. Tween 80 and Triton X-100 were adjusted to 580 μg/mland 90 μg/ml, respectively. The final volume was adjusted to 3 l withphosphate buffered saline. The trivalent pool was filtered ending with0.8 μm cellulose acetate membrane to obtain the trivalent final bulk.Trivalent final bulk was filled into syringes at least 0.5 mL in each.

TABLE 2 Comparison of time dependent HA content measured by SRD intrivalent final bulks which was recovered from syringes. Vaccine 0 2 4 6formul. Strain months months months months Influenza A/NCal/20/99 33 3236 31 vaccine (32-34) (31-33) (34-38) (30-32) without stabilizerA/Pan/2007/99 29 31 34 32 (27-31) (28-34) (32-36) (31-33) B/Yam/166/9836 33 32 31 (34-38) (32-34) (30-34) (29-33) Influenza A/NCal/20/99 31 3236 32 vaccine (30-32) (31-33) (34-38) (31-33) containing alpha-A/Pan/2007/99 33 33 36 33 tocopherol (30-36) (30-36) (35-37) (31-35)succinate B/Yam/166/98 37 36 38 36 (35-39) (34-38) (35-41) (33-39)

Example 3 SRD Method Used to Measure Haemagglutinin Content

Glass plates (12.4-10.0 cm) are coated with an agarose gel containing aconcentration of anti-influenza HA serum that is recommended by NIBSC.After the gel has set, 72 sample wells (3 mm Ø) are punched into theagarose. 10 microliters of appropriate dilutions of the reference andthe sample are loaded in the wells. The plates are incubated for 24hours at room temperature (20 to 25° C.) in a moist chamber. After that,the plates are soaked overnight with NaCl-solution and washed briefly indistilled water. The gel is then pressed and dried. When completely dry,the plates are stained on Coomassie Brillant Blue solution for 10 minand destained twice in a mixture of methanol and acetic acid untilclearly defined stained zones become visible. After drying the plates,the diameter of the stained zones surrounding antigen wells is measuredin two directions at right angles. Alternatively equipment to measurethe surface can be used. Dose-response curves of antigen dilutionsagainst the surface are constructed and the results are calculatedaccording to standard slope-ratio assay methods (Finney, D. J. (1952).Statistical Methods in Biological Assay. London: Griffin, Quoted in:Wood, J M, et al (1977). J. Biol. Standard. 5, 237-247).

Example 4 Clinical Testing of α-Tocopherol Stabilised Influenza Vaccine(Reduced Thiomersal)

Syringes obtained as described in Example 2 are used for clinicaltesting

H3N2: A/Panama/2007/99 RESVIR-17

H1N1: A/New Caledonia/20/99 (H1N1) IVR-116

B: B/Yamanashi/166/98

TABLE 3 thio- thio- Adults 18-60 reduced plus years H3N2 H1N1 B H3N2H1N1 B pre- GMT 47 41 111 55 37 102 vacc. Titer <10 [%] 10.3% 13.8% 1.7%5.3% 12.3% 8.8% Titer ≧40, SPR 60.3% 55.2% 75.9% 70.2% 52.6% 75.4% [%]post- Seroconv. rate 10.3% 13.8% 1.7% 5.3% 12.3% 8.8% vacc. [%]Significant 58.6% 74.1% 58.6% 63.2% 73.7% 52.6% Increase in antibodytiter [%] Seroconversions 58.6% 74.1% 58.6% 63.2% 73.7% 52.6% [%] GMT328 525 766 324 359 588 Fold GMT 7.3 13.0 6.9 5.9 9.8 5.9 Titer ≧40, SPR100.0% 100.0% 100.0% 100.0% 100.0% 100.0% [%] n.d. = C.I. for proportionp = n/N is not defined, because p*(1 − p)*N < 9 n/N = responders (n) aspart of number of subjects of the (sub)population (N), i.e.seroconversions or significant increase, see also: CPMAP/BWP/214/96 12Mar. 1997, p. 17ff GMT = geometric mean titer, reciprocal 95% C.I. = 95%confidence interval, SPR = Seroprotection rate: proportion of subjectswith a protective titer pre- or postvaccination ≧40 titer = HI-antibodytiter Seroconversion rate = proportion of subjects with antibodyincrease from <10 prevaccination to ≧40 postvaccination fold GMT = foldincrease of GMT Significant increase = proportion of subjects with anantibody titer <10 prevaccination and 4-fold antibody increasepostvaccination (two steps of titer) req. = EU requirementSeroconversions = neg to pos or g.e. 4-fold (neg: titer <10, pos: titer≧40) = proportion of subjects with either seroconversion (<10 to ≧40) orsignificant increase.

Results show that the vaccine is able to offer equivalent protection tovaccines containing thiomersal as a preservative.

Example 5a Preparation of Influenza Virus Antigen Preparation Usingα-Tocopherol Succinate as a Stabiliser for a Thiomersal-Free Vaccine

Monovalent split vaccine was prepared according to the followingprocedure.

Preparation of Virus Inoculum

On the day of inoculation of embryonated eggs a fresh inoculum isprepared by mixing the working seed lot with a phosphate buffered salinecontaining gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25μg/ml. (virus strain-dependent). The virus inoculum is kept at 2-8° C.

Inoculation of Embryonated Eggs

Nine to eleven day old embryonated eggs are used for virus replication.Shells are decontaminated. The eggs are inoculated with 0.2 ml of thevirus inoculum. 60,000 inoculated eggs are incubated at the appropriatetemperature (virus strain-dependent) for 48 to 96 hours. At the end ofthe incubation period, the embryos are killed by cooling and the eggsare stored for 12-60 hours at 2-8° C.

Harvest

The allantoic fluid from the chilled embryonated eggs is harvested.Usually, 8 to 10 ml of crude allantoic fluid is collected per egg.

Concentration and Purification of Whole Virus From Allantoic FluidClarification

The harvested allantoic fluid is clarified by moderate speedcentrifugation (range: 4000-14000 g).

Precipitation Step

Saturated ammonium sulfate solution is added to the clarified virus poolto reach a final ammonium sulfate concentration of 0.5 mol/L. Aftersedimentation for at least 1 hour, the precipitate is removed byfiltration on depth filters (typically 0.5 μm)

Filtration

The clarified crude whole virus bulk is filtered on filter membranesending with a validated sterile membrane (typically 0.2 μm).

Ultrafiltration

The sterile filtered crude monovalent whole virus bulk is concentratedon a cassettes equipped with 1000 kDa MWCO BIOMAX™ membrane at least 6fold. The concentrated retentate is washed with phosphate bufferedsaline at least 1.8 times.

Sucrose Gradient Centrifugation

The influenza virus is concentrated by isopycnic centrifugation in alinear sucrose gradient (0.55% (w/v)). The flow rate is 8-15litres/hour.

At the end of the centrifugation, the content of the rotor is recoveredby four different fractions (the sucrose is measured in arefractometer):

fraction 1 55-52% sucrose fraction 2 approximately 52-38% sucrosefraction 3 38-20% sucrose* fraction 4 20-0% sucrose *virusstrain-dependent: fraction 3 can be reduced to 15% sucrose.

For further vaccine preparation, either only fraction 2 is used orfraction 2 together with a further purified fraction 3 are used.

Fraction 3 is washed by diafiltration with phosphate buffer in order toreduce the sucrose content to approximately below 6%. Optionally thisstep may be omitted. The influenza virus present in this dilutedfraction is pelleted to remove soluble contaminants.

The pellet is resuspended and thoroughly mixed to obtain a homogeneoussuspension. Fraction 2 and the resuspended pellet of fraction 3 arepooled and phosphate buffer is added to obtain a volume of approximately40 litres. This product is the monovalent whole virus concentrate.

Sucrose Gradient Centrifugation With Sodium Deoxycholate

The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v) and Tocopherylsuccinate is added for B-strain viruses up to 0.5mM. The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v) andis strain dependent. The flow rate is 8-15 litres/hour.

At the end of the centrifugation, the content of the rotor is recoveredby three different fractions (the sucrose is measured in arefractometer) Fraction 2 is used for further processing. Sucrosecontent for fraction limits (47-18%) varies according to strains and isfixed after evaluation:

Sterile Filtration

The split virus fraction is filtered on filter membranes ending with a0.2 μm membrane. Phosphate buffer containing 0.025% (w/v) Tween 80 and(for B strains) 0.5 mM Tocopherylsuccinate is used for dilution. Thefinal volume of the filtered fraction 2 is 5 times the original fractionvolume.

Inactivation

The filtered monovalent material is incubated at 22±2° C. for at most 84hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffer containing 0.025% (w/v) Tween 80 is thenadded in order to reduce the total protein content down to max. 450μg/ml. For B-strains a phosphate buffered saline containing 0.025% (w/v)Tween 80 and 0.25 mM Tocopherylsuccinate is applied for dilution toreduce the total protein content down to 450 μg/ml. Formaldehyde isadded to a final concentration of 100 μg/ml and the inactivation takesplace at 20° C.±2° C. for at least 72 hours.

Ultrafiltration

The inactivated split virus material is concentrated at least 2 fold ina ultrafiltration unit, equipped with cellulose acetate membranes with20 kDa MWCO. The Material is subsequently washed with phosphate buffercontaining 0.025% (w/v) Tween 80 and following with phosphate bufferedsaline containing 0.01% (w/v) Tween. For B-strain viruses a phosphatebuffered saline containing 0.01% (w/v) Tween 80 and 0.1 mMTocopherylsuccinate is used for washing.

Final Sterile Filtration

The material after ultrafiltration is filtered on filter membranesending with a 0.2 μm membrane. Filter membranes are rinsed and thematerial is diluted if necessary that the protein concentration does notexceed 500 μg/ml with phosphate buffered saline containing 0.01% (w/v)Tween 80 and, specific for B strains, 0.1 mM Tocopherylsuccinate.

Storage

The monovalent final bulk is stored at 2-8° C. for a maximum of 18months.

Stability

TABLE 4 Comparison of time dependent HA content (μg/ml) measured by SRDin monovalent final bulks. After 4 weeks 6 month Strain Stabiliserproduction at 30° C. at 2-8° C. B/Johannesburg/ Tocopherol 214 196 2065/99 succinate (92%) (96%) B/Johannesburg/ None 169 107 153 5/99** (63%)(90%) **produced according to example 1 without Tocopherylsuccinate.

Example 5b Preparation of Influenza Virus Antigen Preparation Usingα-Tocopherol Succinate as a Stabiliser for a Thiomersal-Free Vaccine

A preferred variation of the method described in Example 5a is asfollows:

Harvesting of the whole virus is followed by the precipitation step(ammonium sulfate precipitation). This is followed by the clarificationstep where the fluid is clarified by moderate speed centrifugation(range 4000-14000 g). Thus the order of the precipitation andclarification steps is reversed compared to Example 5a.

Sterile filtration, ultrafiltration and ultracentrifugation (sucrosegradient centrifugation) steps follow as for Example 5a. However, thereis no need for reprocessing step of the fractions resulting from theultracentrifugation step.

The remaining steps in the process are as described in Example 5a.

Thus, the summarised process in this example is as follows:

-   -   Harvest    -   Precipitation (ammonium sulfate)    -   Clarification    -   Sterile filtration    -   Ultrafiltration    -   Ultracentrifugation    -   Splitting (preferably sodium deoxycholate)    -   Sterile filtration    -   Inactivation    -   Ultrafiltration    -   Final sterile filtration

Another preferred variation of Example 5a involves a prefiltration stepbefore the first sterile filtration. This uses a membrane which does notsterile filter but which enables the removal of contaminants e.g.albumin prior to sterile filtration. This can result in a better yield.A suitable membrane for prefiltration is about 0.8 μm to about 1.8 μm,for example 1.2 μm. The prefiltration step can be used in the scheme ofExample 5a or Example 5b.

Example 6 Preparation of Influenza Vaccine Using α-Tocopherol Succinateas a WStabiliser for a Thiomersal-Free Vaccine

Monovalent final bulks of three strains, A/New Caldonia/20/99 (H1N1)IVR-116, A/Panama/2007/99 (H3N2) Resvir-17 and B/Yamanashi/166/98 wereproduced according to the method described in Example 5.

Pooling

The appropriate amount of monovalent final bulks was pooled to a finalHA-concentration of 30 μg/ml for A/New Caldonia/20/99 (H1N1) IVR-116,A/Panama/2007/99 (H3N2) Resvir-17, respectively and of 36 μg/ml forB/Johannesburg/5/97. Tween 80 and Triton X-100 were adjusted to 580μg/ml and 90 μg/ml, respectively. The final volume was adjusted to 3 lwith phosphate buffered saline. The trivalent pool was filtered endingwith 0.8 μm cellulose acetate membrane to obtain the trivalent finalbulk. Trivalent final bulk was filled into syringes at least 0.5 mL ineach.

TABLE 5 Comparison of time dependent HA content (μg/ml) measured by SRDin trivaient final bulks. 0 4 weeks 6 months Vaccine formul. Strainmonths at 30° C. at 2-8° C. Influenza vaccine A/NCal/20/99 31 32 30without stabilizer A/Pan/2007/99 31 34 33 B/Joh/5/99* 35 25 31 Influenzavaccine A/NCal/20/99 34 35 34 containing alpha-tocopherol A/Pan/2007/9933 33 34 succinate B/Joh/5/99** 29 25 28 *Formulation was based ontarget concentration of 39 μg/ml. **Formulation was based on targetconcentration of 34 μg/ml.

Example 7 Preparation of Influenza Virus Antigen Preparation UsingSodium Lauryl Sulfate as a Stabiliser for a Preservative-Free Vaccine(Thiomersal-Reduced Vaccine) Monovalent Whole Virus Concentrate ofB/Johannesburg/5/99 was Obtained as Described in Example 1. SucroseGradient Centrifugation With Sodium Deoxycholate

The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v). The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v)and is strain dependent. The flow rate is 8-15 litres/hour. At the endof the centrifugation, the content of the rotor is recovered by threedifferent fractions (the sucrose is measured in a refractometer)Fraction 2 is used for further processing. Sucrose content for fractionlimits (47-18%) varies according to strains and is fixed afterevaluation:

Sterile Filtration

A sample of fraction 2 of 10 ml was taken for further processing. Thesplit virus fraction is filtered on filter membranes ending with a 0.2μm membrane. Phosphate buffer containing 0.025% (w/v) Tween 80 and 0.5mM sodium lauryl sulfate is used for dilution. The final volume of thefiltered fraction 2 is 5 times the original fraction volume.

Inactivation

The filtered monovalent material is incubated at 22±2° C. for at most 84hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffered saline containing 0.025% (w/v) Tween 80and 0.5 mM sodium laurylsulfate is then added in order to reduce thetotal protein content down to max. 250 μg/ml. Formaldehyde is added to afinal concentration of 50 μg/ml and the inactivation takes place at 20°C.±2° C. for at least 72 hours.

Ultrafiltration

The inactivated split virus material is concentrated at least 2 fold ina ultrafiltration unit, equipped with cellulose acetate membranes with20 kDa MWCO. The Material is subsequently washed with 4 volumesphosphate buffered saline containing 0.01% (w/v) Tween and 0.5 mM sodiumlauryl sulfate.

Final Sterile Filtration

The material after ultrafiltration is filtered on filter membranesending with a 0.2 μm membrane. Filter membranes are rinsed and thematerial is diluted if necessary that the protein concentration does notexceed 500 μg/ml with phosphate buffered saline containing 0.01% (w/v)Tween 80 and 0.5 mM sodium lauryl sulfate.

Storage

The monovalent final bulk is stored at 2-8° C.

TABLE 6 Comparison of time dependent HA content measured by SRD inmonovalent final bulks. 4 weeks stabiliser After production at 30° C.B/Johannesburg/5/99 None* 182 139 (77%) B/Johannesburq/5/99 Sodiumlauryl 288 264 (92%) sulfate *produced according to Example 7 withoutaddition of sodium lauryl sulfate

Example 8 Preparation of Influenza Virus Antigen Preparation UsingPlantacare or Laureth-9 as a Stabiliser for a Preservative-Free Vaccine(Thiomersal-Reduced Vaccine) Monovalent Whole Virus Concentrate ofB/Yamanashi/166/98 was Obtained as Described in Example 1. Fragmentation

The monovalent whole influenza virus concentrate is diluted to a proteinconcentration of 1,000 μg/ml with phosphate buffered saline pH 7.4.Either Plantacare® 2000 UP or Laureth-9 is added to a finalconcentration of 1% (w/v). The material is slightly mixed for 30 min.Then the material is overlayed on a sucrose cushion 15% (w/w) in abucket. Ultracentrifugation in a Beckman swing out rotor SW 28 isperformed for 2 h at 25,000 rpm and 20° C.

Sterile Filtration

A supernatant was taken for further processing. The split virus fractionis filtered on filter membranes ending with a 0.2 μm membrane.

Inactivation

Phosphate buffered saline is added if necessary in order to reduce thetotal protein content down to max. 500 μg/ml. Formaldehyde is added to afinal concentration of 100 μg/ml and the inactivation takes place at 20°C.±2° C. for at least 6 days.

Ultrafiltration

Tween 80 and Triton X 100 is adjusted in the inactivated material to0.15% and 0.02% respectively. The inactivated split virus material isconcentrated at least 2 fold in a ultrafiltration unit, equipped withcellulose acetate membranes with 30 kDa MWCO. The Material issubsequently washed with 4 volumes phosphate buffered saline.

Final Sterile Filtration

The material after ultrafiltration is filtered on filter membranesending with a 0.2 μm membrane. Filter membranes are rinsed and thematerial is diluted that the protein concentration does not exceed 500μg/ml with phosphate buffered saline

Storage

The monovalent final bulk is stored at 2-8° C.

TABLE 7 Comparison of time dependent HA content measured by SRD inmonovalent final bulks. 4 weeks stabiliser After production at 30° C.B/Yamanashi/166/98 None 143 98 (68%) B/Yamanashi/166/98 Plantacare ® 476477 (100%) 2000 UP B/Yamanashi/166/98 Laureth-9 468  494 (>100%)

Example 9 Clinical Testing of α-Tocopherol Stabilised Influenza Vaccine(Reduced Thiomersal) in the Elderly via ID and IM Administration APreparation of Influenza Virus Antigen Preparation

Monovalent split vaccine was prepared according to the followingprocedure.

Preparation of Virus Inoculum

On the day of inoculation of embryonated eggs a fresh inoculum isprepared by mixing the working seed lot with a phosphate buffered salinecontaining gentamycin sulphate at 0.5 mg/ml and hydrocortisone at 25μg/ml. (virus strain-dependent). The virus inoculum is kept at 2-8° C.

Inoculation of Embryonated Eggs

Nine to eleven day old embryonated eggs are used for virus replication.Shells are decontaminated. The eggs are inoculated with 0.2 ml of thevirus inoculum. The inoculated eggs are incubated at the appropriatetemperature (virus strain-dependent) for 48 to 96 hours. At the end ofthe incubation period, the embryos are killed by cooling and the eggsare stored for 12-60 hours at 2-8° C.

Harvest

The allantoic fluid from the chilled embryonated eggs is harvested.Usually, 8 to 10 ml of crude allantoic fluid is collected per egg.

Concentration and Purification of Whole Virus From Allantoic Fluid

1. Clarification

The harvested allantoic fluid is clarified by moderate speedcentrifugation (range: 4000-14000 g).

2. Adsorption Step

To obtain a CaHPO₄ gel in the clarified virus pool, 0.5 mol/L Na₂HPO₄and 0.5 mol/L CaCl₂ solutions are added to reach a final concentrationof CaHPO₄ of 1.5 g to 3.5 g CaHPO₄/litre depending on the virus strain.

After sedimentation for at last 8 hours, the supernatant is removed andthe sediment containing the influenza virus is resolubilised by additionof a 0.26 mol/L EDTA-Na₂ solution, dependent on the amount of CaHPO₄used.

3. Filtration

The resuspended sediment is filtered on a 6 μm filter membrane.

4. Sucrose Gradient Centrifugation

The influenza virus is concentrated by isopycnic centrifugation in alinear sucrose gradient (0.55% (w/v)) containing 100 μg/ml Thiomersal.The flow rate is 8-15 litres/hour.

At the end of the centrifugation, the content of the rotor is recoveredby four different fractions (the sucrose is measured in arefractometer):

fraction 1 55-52% sucrose fraction 2 approximately 52-38% sucrosefraction 3 38-20% sucrose* fraction 4 20-0% sucrose *virusstrain-dependent: fraction 3 can be reduced to 15% sucrose.

For further vaccine preparation, only fractions 2 and 3 are used.

Fraction 3 is washed by diafiltration with phosphate buffer in order toreduce the sucrose content to approximately below 6%. The influenzavirus present in this diluted fraction is pelleted to remove solublecontaminants.

The pellet is resuspended and thoroughly mixed to obtain a homogeneoussuspension. Fraction 2 and the resuspended pellet of fraction 3 arepooled and phosphate buffer is added to obtain a volume of approximately40 litres, a volume appropriate for 120,000 eggs/batch. This product isthe monovalent whole virus concentrate.

5. Sucrose Gradient Centrifugation With Sodium Deoxycholate

The monovalent whole influenza virus concentrate is applied to aENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrosegradient (0.55% (w/v)) where a sodium deoxycholate gradient isadditionally overlayed. Tween 80 is present during splitting up to 0.1%(w/v) and Tocopherol succinate is added for B-strain-viruses up to 0.5mM. The maximal sodium deoxycholate concentration is 0.7-1.5% (w/v) andis strain dependent. The flow rate is 8-15 litres/hour.

At the end of the centrifugation, the content of the rotor is recoveredby three different fractions (the sucrose is measured in arefractometer) Fraction 2 is used for further processing. Sucrosecontent for fraction limits (47-18%) varies according to strains and isfixed after evaluation:

6. Sterile Filtration

The split virus fraction is filtered on filter membranes ending with a0.2 μm membrane. Phosphate buffer containing 0.025% (w/v) Tween 80 and(for B strain viruses) 0.5 mM Tocopherol succinate is used for dilution.The final volume of the filtered fraction 2 is 5 times the originalfraction volume.

7. Inactivation

The filtered monovalent material is incubated at 22±2° C. for at most 84hours (dependent on the virus strains, this incubation can beshortened). Phosphate buffer containing 0.025% (w/v). Tween 80 is thenadded in order to reduce the total protein content down to max. 250μg/ml. For B strain viruses, a phosphate buffered saline containing0.025% (w/v) Tween 80 and 0.25 mM Tocopherol succinate is applied fordilution to reduce the total protein content down to 250 μg/ml.Formaldehyde is added to a final concentration of 50 μg/ml and theinactivation takes place at 20° C.±2° C. for at least 72 hours.

8. Ultrafiltration

The inactivated split virus material is concentrated at least 2 fold ina ultrafiltration unit, equipped with cellulose acetate membranes with20 kDa MWCO. The Material is subsequently washed with phosphate buffercontaining 0.025% (w/v) Tween 80 and following with phosphate bufferedsaline containing 0.01% (w/v) Tween. For B strain virus a phosphatebuffered saline containing 0.01% (w/v) Tween 80 and 0.1 mM Tocopherolsuccinate is used for washing.

9. Final Sterile Filtration

The material after ultrafiltration is filtered on filter membranesending with a 0.2 μm membrane. Filter membranes are rinsed and thematerial is diluted if necessary such that the protein concentrationdoes not exceed 1,000 μg/ml but haemagglutinin concentration exceeds 180μg/ml with phosphate buffered saline containing 0.01% (w/v) Tween 80 and(for B strain viruses) 0.1 mM Tocopherol succinate.

10. Storage

The monovalent final bulk is stored at 2-8° C. for a maximum of 18months.

B Preparation of Influenza Vaccine

Monovalent final bulks of three strains, A/New Caldonia/20/99 (H1N1)IVR-116, A/Panama/2007/99 (H3N2) Resvir-17 and B/Johannesburg/5/99 wereproduced according to the method described in part A above.

Pooling

The appropriate amount of monovalent final bulks was pooled to a finalHA-concentration of 60 μg/ml for A/New Caldonia/20/99 (H1N1) IVR-116,A/Panama/2007/99 (H3N2) Resvir-17, respectively and of 68 μg/ml forB/Johannesburg/5/99. Tween 80, Triton X-100 and Tocopherol succinatewere adjusted to 1,000 μg/ml, 110 μg/ml and 160 μg/ml, respectively. Thefinal volume was adjusted to 3 with phosphate buffered saline. Thetrivalent pool was filtered ending with 0.8 μm cellulose acetatemembrane to obtain the trivalent final bulk. Trivalent final bulk wasfilled into syringes at least 0.165 mL in each.

Vaccine Administration

The vaccine was supplied in pre-filled syringes and was administeredintradermally in the deltoid region. The intradermal (ID) needle was asdescribed in EP1092444, having a skin penetration limiter to ensureproper intradermal injection. Since formation of a wheal (papule) at theinjection site demonstrates the good quality of ID administration, theinvestigator with the subject measured the exact size of the wheal 30minutes after vaccination.

One dose (100 μl) contained the following components:

HEMAGGLUTININ FROM THREE INFLUENZA STRAINS A/NEW CALEDONIA/20/99 6.0 μgA/PANAMA/2007/99 6.0 μg B/JOHANNESBURG 5/99 6.0 μg THIOMERSALPRESERVATIVE 0.4 μg-0.8 μg

B The Above Vaccine Was Compared a Standard Trivalent Split InfluenzaVaccine:

Fluarix™. The Fluarix vaccine was supplied in pre-filled syringes andwas administered intramuscularly in the deltoid muscle. A needle of atleast 2.5 cm/1 inch in length (23 gauge) was used to ensure properintramuscular injection.

One dose (0.5 ml) contained the following components:

HEMAGGLUTININ FROM THREE INFLUENZA STRAINS A/NEW CALEDONIA/20/99 15.0 μgA/PANAMA/2007/99 15.0 μg B/JOHANNESBURG 5/99 15.0 μg THIOMERSALPRESERVATIVE 50.0 μg

Results

The mean age of the total cohort at the time of vaccine administrationwas 70.4±6.2 years Standard Deviation (S.D.), the female/male ratio was1.7:1.

Immunogenicity results: Analysis of derived immunogenicity variables wasas follows: Variable Flu-red ID (N = 65) Fluarix ™ IM (N = 65) GMT GMTLL UL GMT LL UL A/NEW CALEDONIA PRE 99.5 76.9 128.7 90.0 70.1 115.7 POST165.1 129.2 211.0 174.3 133.3 227.9 A/PANAMA PRE 75.5 54.7 104.2 69.251.9 92.4 POST 128.6 99.1 166.8 164.3 126.0 214.1 B/JOHANNESBURG PRE236.0 187.7 296.8 222.6 176.9 280.2 POST 341.2 276.0 421.7 402.4 312.1518.9 Seroconversion rate % LL UL % LL UL A/NEW CALEDONIA 15.4 7.6 26.518.5 9.9 30.0 A/PANAMA 20.0 11.1 31.8 29.2 18.6 41.8 B/JOHANNESBURG 9.23.5 19.0 16.9 8.8 28.3 Conversion factor GMR LL UL GMR LL UL A/NEWCALEDONIA 1.7 1.4 2.0 1.9 1.6 2.3 A/PANAMA 1.7 1.4 2.1 2.4 1.9 3.0B/JOHANNESBURG 1.4 1.2 1.7 1.8 1.5 2.1 Seroprotection rate % LL UL % LLUL A/NEW CALEDONIA PRE 87.7 77.2 94.5 90.8 81.0 96.5 POST 92.3 83.0 97.596.9 89.3 99.6 A/PANAMA PRE 75.4 63.1 85.2 81.5 70.0 90.1 POST 90.8 81.096.5 93.8 85.0 98.3 B/JOHANNESBURG PRE 98.5 91.7 100.0 96.9 89.3 99.6POST 100.0 94.5 100.0 98.5 91.7 100.0 N: number of subjects withavailable results; %: percentage of subjects within the given parameter;LL/UL: lower and upper limit of 95% Cl; Pre: at the time of vaccineadministration; Post: 21 days after the vaccine dose

Injection site pain, reported by 10/65 (15.4%) vaccinees, was the mostcommon symptom following IM administration of Fluarix™. In the ID group,pain was reported by 3/65 (4.6%) vaccinees. This difference wasstatistically significant (p=0.038; Fisher exact test). Accordingly theID delivery of a thiomersal reduced product is preferred.

Conclusions

Both ID and IM administration of a thio-reduced flu vaccine in anelderly population can provide 100% seroprotection.

A comparable response to vaccination in terms of geometric mean titers,seroprotection rates, seroconversion rates and conversion factors wasfound in IM and ID vaccinated individuals where the ID group received2.5-fold less antigen. There was no discernible difference in theoverall incidence of vaccine-related solicited/unsolicited systemicsymptoms in the two treatment groups.

Example 10 Intradermal Delivery of a Thiomersal-Reduced InfluenzaVaccine

Immunogenicity of the thiomersal reduced split influenza vaccineprepared as described in Example 9 (except that the pooling was doneindependently and the vaccine was not filled into syringes) was assessedby ID delivery in guinea pigs using a standard needle.

Groups of 5 animals each were primed intranasally with whole inactivatedtrivalent influenza virus containing 5 μg of each HA in a total volumeof 200 μl. Twenty-eight days after priming the animals were vaccinatedby either the intradermal or intramuscular routes. Intradermal dosescontaining 0.1, 0.3, or 1.0 μg trivalent thiomersal-reduced split Flu in0.1 ml were administered in the back of the guinea pig using a standardneedle An intramuscular dose of 1.0 μg trivalent thiomersal-reducedsplit Flu was administered in the hind leg of the guinea pig in a volumeof 0.1 ml. The groups were as follows:

-   Group 1—0.1 μg trivalent thiomersal-reduced split Flu ID;-   Group 2—0.3 μg trivalent thiomersal-reduced split Flu ID;-   Group 3—1.0 μg trivalent thiomersal-reduced split Flu ID-   Group 4—1.0 μg trivalent thiomersal-reduced split Flu IM

Fourteen days after vaccination the animals were bled and the antibodytiters induced by the vaccination were assessed using a standardhemagglutination inhibition assay (HI). The results are shown in FIG. 1.Strong HI responses to all three strains were induced by vaccination. Noclear dose response was noted suggesting that very low doses ofthiomersal-reduced antigen can still induce very potent HI antibodyresponses when administered by the ID route. There was no significantdifference between the HI titers induced by ID or IM vaccination. Thus,the results obtained in guinea pigs confirmed that thethimerosal-reduced trivalent split influenza antigens induce similarlevels of HI antibodies in animals when delivered by the ID routecompared to the IM route.

Example 11 Intradermal Delivery of a Thiomersal-Reduced, AdjuvantedInfluenza Vaccine Protocol

Guinea pigs were primed on Day 0 with 5 μg trivalent whole inactivatedFlu virus in 200 μl, intranasally.

Vaccination—Day 28—Vaccine containing 0.1 μg HA per strain trivalentsplit Flu prepared as described in Example 9 (except that the poolingstep resulted in a final concentration for each antigen of 1.0 μg/ml togive a dose of 0.1 μg in 100 μl compared to 60 μg/ml in Example 9). Thefinal trivalent formulation was administered intradermally usingtuberculin syringes, either adjuvanted or unadjuvanted, in 100 μl.

Bleeding—Day 42.

The effect of adjuvantation was assessed by measuring antibody responsesby HI assay (day 0, 28, 42).

All ID experiments were carried out using a standard needle.

Results

G1-G5 refer to 5 groups of guinea pigs, 5 per group.

G1 Split trivalent thiomersal reduced 0.1 μg

G2 Split trivalent thio red 0.1 μg+3D-MPL 50 μg

G3 Split trivalent thio red 0.1 μg+3D-MPL 10 μg

G4 Split trivalent thio red 0.1 μg+3D-MPLin 50 μg+QS21 50 μg

G5 Split trivalent thio red 0.1 μg+3D-MPLin 10 μg+QS21 10 μg

Note 3D-MPLin+QS21 refers to an adjuvant formulation which comprises aunilamellar vesicle comprising cholesterol, having a lipid bilayercomprising dioleoyl phosphatidyl choline, wherein the QS21 and the3D-MPL are associated with, or embedded within, the lipid bilayer. Suchadjuvant formulations are described in EP 0 822 831 B, the disclosure ofwhich is incorporated herein by reference.

HI Titres anti-A/New Caledonia/20/99

NC Pre-immun Pre-boost Post-boost G1 5 10 92 G2 5 10 70 G3 5 11 121 G4 79 368 G5 5 10 243

HI Titres anti-A/Panama/2007/99

P Pre-immun Pre-boost Post-boost G1 5 485 7760 G2 5 279 7760 G3 5 4858914 G4 7 485 47051 G5 5 320 17829

HI Titres anti-B/Johannesburg/5/99

J Pre-immun Pre-boost Post-boost G1 5 23 184 G2 5 11 121 G3 5 11 70 G4 615 557 G5 5 13 320

Thus, whether adjuvanted or unadjuvanted the thiomersal-reducedtrivalent split Flu antigen is a potent immunogen and capable ofinducing strong HI responses when administered by the ID or IM route.These responses appear to be at least as potent as the responses inducedby the standard Fluarix preparation.

Example 12 Comparison of Thiomersal-Containing and Thiomersal-FreeVaccine Delivered Intradermally in Pigs

In order to assess the immunogenicity of the split Flu vaccine (plus andminus thiomersal) administered by the ID route the primed pig model wasused. As the vast majority of the population has experienced at leastone infection with influenza an influenza vaccine must be able to boosta pre-existing immune response. Therefore animals are primed in aneffort to best simulate the human situation.

In this experiment 4 week old pigs were primed by the intranasal route.Six groups of five animals each were primed as follows:

Group 1—two primings of trivalent whole inactivated virus (50 μg eachHA) at day 0 and 14; Group 2—two primings of trivalent whole inactivatedvirus (50 μg each HA) at day 0 and 14; Group 3—single priming withtrivalent whole inactivated virus (50 μg each HA) at day 0; Group 4—twoprimings of trivalent whole inactivated virus (25 μg each HA) at day 0and 14; Group 5—single priming of trivalent whole inactivated virus (25μg each HA) at day 0; Group 6—two primings of trivalent wholeinactivated virus (12.5 μg each HA) at day 0 and 14.

On day 28 post final priming, the animals were vaccinated with 3 μg eachHA trivalent split antigen (strains A/New Caledonia H1N1, A/Panama H3N2,and B/Johannesburg) in 100 μl by the ID route. Group 1 received standardFluarix™ containing thiomersal preservative as vaccine antigen. Allother groups received the preservative-free antigen.

The HI results obtained in this experiment are shown in FIG. 2(Anti-Influenza Hemagglutination Inhibition Titers Induced in PigsPrimed with a Variety of Antigen Doses and Vaccinated with 3 MicrogramsTrivalent Influenza Antigen Plus or Minus Thiomersal by the IntradermalRoute).

Relatively low HI titers are induced to the B strain in this experimentand the background against the A/H3N2 strain is high. A beneficialeffect in terms of response to vaccination is observed when the primingdose is reduced. In almost all cases, reduction in the antigenconcentration or number of priming doses (from the two primings with 50μg) resulted in a heightened response to vaccination. While the responseof the animals in Groups 1 and 2, which were primed twice with 50 μg, tovaccination is not so evident, it appears that the preservative-freeantigen (Group 2) functions at least as well as Fluarix™ (Group 1) underthese conditions. A strong response to vaccination withpreservative-free trivalent influenza antigen administered by the IDroute in the alternatively primed animals (Groups 3-6) is clear and thisresponse is seen even in the B strain, although the HI titers remainlow.

1-35. (canceled)
 36. An influenza vaccine comprising an inactivatedinfluenza virus preparation and an adjuvant, wherein said inactivatedinfluenza virus preparation comprises hemagglutinin antigen (HA), 0μg/ml to 5 μg/ml of thiomersal and at least one stabilising excipient,and wherein said adjuvant is selected from the group of: an oil in wateremulsion, a non-toxic derivative of lipid A, a saponin or derivativethereof, and a combination of two or more of said adjuvants.
 37. Theinfluenza vaccine according to claim 36, wherein said non-toxicderivative of lipid A is 3D-MPL.
 38. The influenza vaccine according toclaim 36, wherein 3D-MPL is in the form of an emulsion having a smallparticle size less than 0.2 μm in diameter.
 39. The influenza vaccineaccording to claim 36, wherein said said excipient comprisesα-tocopherol or a derivative thereof.
 40. The influenza vaccineaccording to claim 36, wherein said excipient comprises α-tocopherolsuccinate.
 41. The influenza vaccine according to claim 36, wherein saidexcipient comprises a positively or negatively or zwitterionic chargedamphiphilic molecule.
 42. The influenza vaccine according to claim 36,wherein said excipient comprises a non-ionic amphiphilic molecule fromthe group of: octyl- or nonylphenoxy polyoxyethanols, polyoxyethylenesorbitan esters and polyoxyethylene ethers or esters of general formula(I):HO(CH2CH2O)_(n)-A-R   (I) wherein n is 1-50, A is a bond or —C(O)—, R isC₁₋₅₀ alkyl or phenyl C₁₋₅₀ alkyl; and combinations of two or more ofthese.
 43. The influenza vaccine according to claim 42, wherein saidnon-ionic amphiphilic molecule is selected from the group of: TritonX-45, t-octylphenoxy polyethoxyethanol (Triton X-100™), Triton X-102,Triton X-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57,Triton N-101, Triton N-128, Breij 35, polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-stearyl ether (steareth 9),polyoxyethylene sorbitan ester, and polyoxyethylene sorbitan monooleate(Tween 80™).
 44. The influenza vaccine according to claim 42, whereinsaid non-ionic amphiphilic molecule is selected from the group: TritonX-100™, Tween 80™, and a combination of both.
 45. The influenza vaccineaccording to claim 39, wherein the a-tocopherol or derivative thereof ispresent at a concentration between 1 μg/ml and 10 mg/ml.
 46. Theinfluenza vaccine according to claim 36, wherein the α-tocopherol ispresent at a concentration between 1 μg/ml and 10 mg/ml.
 47. Theinfluenza vaccine according to claim 36, wherein said influenza virusantigen preparation is selected from the group consisting of: splitvirus antigen preparations, subunit antigens, chemically or otherwiseinactivated whole virus and recombinantly produced haemagglutininantigen.
 48. The influenza vaccine according to claim 36, wherein theconcentration of haemagglutinin antigen for each strain of influenza is1-1000 μg per ml, as measured by a Single radial Immunodiffusion (SRD)assay.
 49. The influenza vaccine according to claim 36, wherein saidinfluenza virus antigen preparation is derived from the embryonated eggmethod or derived from cell culture.
 50. The influenza vaccine accordingto claim 36, wherein said influenza virus antigen preparation comprisesa haemagglutinin antigen that is produced recombinantly.
 51. Aninactivated influenza virus preparation, comprising hemagglutininantigen (HA), 0 μg/ml to 5 pg/ml of thiomersal, and at least one ofα-tocopherol or a derivative thereof in an amount sufficient tostabilize said HA.
 52. The inactivated influenza virus preparation ofclaim 50, wherein said influenza virus is selected from the group of:split virus antigen preparations, subunit antigens, chemically orotherwise inactivated whole virus, and recombinantly producedhaemagglutinin antigen.
 53. The inactivated influenza virus preparationof claim 50, wherein said preparation comprises α-tocopherol orα-tocopherol succinate.
 54. The inactivated influenza virus preparationof claim 50, wherein the at least one of α-tocopherol or a derivativethereof is present in an amount such that the HA of said preparationremains stable for at least 6 months after said preparation is producedas determined by the presence of an amount of HA detectable by Singleradial Immunodiffusion (SRD) assay.
 55. The inactivated influenza viruspreparation of claim 50, wherein the at least one of a-tocopherol or aderivative thereof further comprises at least one of a non-ionicamphiphilic molecule from the group of: octyl- or nonylphenoxypolyoxyethanols, polyoxyethylene sorbitan esters and polyoxyethyleneethers or esters of general formula (I):HO(CH2CH2O)_(n)-A-R   (I) wherein n is 1-50, A is a bond or —C(O)—, R isC₁₋₅₀ alkyl or phenyl C₁₋₅₀ alkyl; and combinations of two or more ofthese.
 56. The inactivated influenza virus preparation of claim 55,wherein said non-ionic amphiphilic molecule is selected from the groupof: Triton X-45, t-octylphenoxy polyethoxyethanol (Triton X-100™),Triton X-102, Triton X-114, Triton X-165, Triton X-205, Triton X-305,Triton N-57, Triton N-101, Triton N-128, Breij 35,polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-stearylether (steareth 9), polyoxyethylene sorbitan ester, and polyoxyethylenesorbitan monooleate (Tween 80™).
 57. The inactivated influenza viruspreparation of claim 55, wherein said non-ionic amphiphilic molecule isselected from the group: Triton X-100™, Tween 80™, and a combination ofboth.
 58. The inactivated influenza virus preparation according to claim50, wherein the α-tocopherol or derivative thereof is present at aconcentration between 1 pg/ml and 10 mg/ml.
 59. A vaccine comprising theinactivated influenza virus preparation of claim
 50. 60. An influenzavaccine according to claim 58, wherein the vaccine additionallycomprises an adjuvant.